Ifbm&#39;s to promote the specific attachment of target analytes to the surface of orthopedic implants

ABSTRACT

The present invention provides an improved coating for surfaces of medical implants. The coating comprises at least one interfacial biomaterial (IFBM) which is comprised of at least one binding module that binds to the surface of an implant or implant-related material (“implant module”) and at least one binding module that selectively binds to a target analyte or that is designed to have a desired effect (“analyte module”). The modules are connected by a linker. In some embodiments, the IFBM coating acts to promote the recognition and attachment of target analytes to surface of the device. The IFBM coating improves the performance of implanted medical devices, for example, by promoting osteointegration of the implant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of pending U.S.Non-provisional application Ser. No. 12/871,194, filed on Aug. 30, 2010,which is a divisional application of U.S. Non-provisional applicationSer. No. 12/488,183, filed on Jun. 19, 2009, now U.S. Pat. No.7,812,119, which is a divisional application of U.S. Non-provisionalapplication Ser. No. 11/152,974, filed on Jun. 15, 2005, now U.S. Pat.No. 7,572,766, which claims priority to U.S. Provisional Application No.60/580,019, filed Jun. 16, 2004; U.S. Provisional Application No.60/651,338, filed Feb. 9, 2005; and U.S. Provisional Application No.60/651,747, filed Feb. 10, 2005; each of which is hereby incorporated inits entirety by reference herein.

FIELD OF THE INVENTION

The present invention provides materials and methods for coatingsurfaces of medical devices with interfacial biomaterials that promotethe specific recognition and attachment of the target analyte to thesurface of the device.

BACKGROUND OF THE INVENTION

Orthopedic implants are used for a variety of joint replacements and topromote bone repair in humans and animals. According to medical industryanalysts, there are now over 800,000 hip and knee joint replacementsperformed in human patients each year in the U.S. In addition, hundredsof thousands of human patients undergo surgical procedures in whichorthopedic implants are used, for example, to treat various types ofbone fractures or to relieve severe back pain.

With all of these procedures, there is a need for controlled, directed,rapid healing. Individuals undergoing joint replacement often experienceuncomplicated healing and restoration of function. Unfortunately, thereis a high rate of complications, including “late failures.” The revisionsurgery rate for human total joint replacement varies between 10 to 20%(Malchau et al. (2002) “Prognosis of total hip replacement: Update ofresults and risk-ratio analysis for revision and re-revision from theSwedish National Hip Arthroplasty Registry, 1979-2000,” scientificexhibition at the 69th Annual Meeting of the American Academy ofOrthopaedic Surgeons, Dallas, Tex., Feb. 13-17, 2002; Fitzpatrick et al.(1998) Health Technol. Assess. 2:1-64; Mahomed et al. (2003) J. BoneJoint Surg. Am. 85-A:27-32)). The majority of these revision surgeriesare made necessary by failure at the implant-bone interface.

Orthopedic implants are made of materials which are relatively inert(“alloplastic” materials), typically metallic, ceramic, or plasticmaterials. Previous approaches to improve the outcomes of orthopedicimplant surgeries have mainly focused on physical changes to the implantsurface that result in increased bone formation. These approachesinclude using implants with porous metallic surfaces to promote boneingrowth and spraying implants with hydroxyapatite plasma. Approachesusing dental implants have also included the use oftopographically-enhanced titanium surfaces in which surface roughness isimparted by a method such as grit blasting, acid etching, or oxidation.While these techniques have improved the outcomes of orthopedic implantsurgeries, there is still considerable room for further improvement.

Tissue response to an alloplastic material is known to be influenced bycell adhesion to the material's surface, and much research has beendirected to improving cell adhesion to alloplastic materials. Celladhesion between cells in vivo is known to be controlled primarily bythe binding of short, exposed protein domains in the extracellularmatrix to cell surface receptors (LeBaron & Athanasiou (2000) TissueEng. 6: 85-103; Yamada (1997) Matrix Biol. 16: 137-141). Notably, aclass of receptors known as integrins has been implicated in celladhesion to implant surfaces. Integrins and their target ligands havebeen shown to stimulate osteoblast adhesion and proliferation as well asbone formation (see, e.g., Kantlehner et al. (2000) ChemBioChem 1:107-114; Sarmento et al. (2004) J. Biomed. Mater. Res. 69A: 351-358;Hayashibara et al. (2004) J. Bone Mineral Res. 19: 455-462. Integrinsmay be useful in targeting cell adhesion to implants and in this mannermay improve integration of implants into adjacent bone.

Other research has shown that the local expression of growth factors andcytokines can enhance tissue reactions at alloplastic implant surfaces.For example, Cole et. al. ((1997) Clin. Orthop. 345: 219-228) have shownthat growth factors can promote the integration of an implant intoadjacent bone (“osteointegration”) as well as increase the rate of boneformation next to the implant surface. See also U.S. Pat. No. 5,344,654.

Growth factors that stimulate new bone production (“osteoinductiveproteins”) include, but are not limited to, platelet-derived growthfactor (PDGF), insulin-like growth factors 1 and 2 (IGF-1 and IGF-2),vascular endothelial growth factor (VEGF), fibroblast growth factor(FGF), transforming growth factor (TGF-β), bone morphogenic proteins(BMP), and associated family members.

The most effective osteoinductive proteins are the bone morphogeneticproteins (BMPs). The BMPs are members of the TGF-β superfamily thatshare a set of conserved cysteine residues and a high level of sequenceidentity overall. Over 15 different BMPs have been identified, and mostBMPs stimulate the cascade of events that lead to new bone formation(see U.S. Pat. Nos. 5,013,649; 5,635,373; 5,652,118; and 5,714,589; alsoreviewed by Reddi and Cunningham (1993) J. Bone Miner. Res. 8 Supp. 2:S499-S502; Issack and DiCesare (2003) Am. J. Orthop. 32: 429-436; andSykaras & Opperman (2003) J. Oral Sci. 45: 57-73). This cascade ofevents that leads to new bone formation includes the migration ofmesenchymal stem cells, the deposition of osteoconductive matrix, theproliferation of osteoprogenitor cells, and the differentiation ofprogenitor cells into bone-producing cells. Much research has beendirected to the use of BMPs on or near implants in order to promoteosteointegration of the implants (see, e.g.: Friedlander et al. (2001)J. Bone Joint Surg. Am. 83-A Suppl. 1 (Pt. 2): S151-58; Einhorn (2003)J. Bone Joint Surg. Am. 85-A Suppl. 3: 82-88; Burkus et al. (2002) J.Spinal Disord. Tech. 15(5): 337-49). However, one of the critical issuesthat remains unresolved is the method of grafting or immobilizing anactive BMP or other active biomolecule onto the surface of an implant.

It has been shown that the presentation of BMPs is critical forproducing desired bone formation next to an implant device. Approachesto improving implants have been modeled in view of the natural processof bone formation. In human bone, collagen serves both as a scaffold forbone formation and as a natural carrier for BMPs. Demineralized bone hasbeen used successfully as a bone graft material; the main components ofdemineralized bone are collagen and BMPs (see U.S. Pat. No. 5,236,456).Many matrix systems have been developed that are designed to encouragebone formation by steadily releasing growth factors and other bioactivemolecules as the matrix degrades. The efficiency of BMP release frompolymer matrixes depends on matrix characteristics such as the affinityof BMP for the matrix, resorbtion rate, density, and pore size.Materials used in such matrix systems include organic polymers whichreadily hydrolyze in the body into inert monomers. Such organic polymersinclude polylactides, polyglycolides, polyanhydrides, andpolyorthoesters (see U.S. Pat. Nos. 4,563,489; 5,629,009; and4,526,909). Other materials described as being useful in BMP-containingmatrices include polylactic and polyglycolic acid copolymers, alginate,poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer, andpoly (vinyl alcohol) (see U.S. Pat. No. 5,597,897). Natural matrixproteins have also been used to deliver BMPs to bone areas; thesenatural proteins include collagen, glycosaminoglycans, and hyaluronicacid, which are enzymatically digested in the body (see U.S. Pat. Nos.4,394,320; 4,472,840; 5,366,509; 5,606,019; 5,645,591; and 5,683,459).

Even with the use of a polymer matrix to retain BMP at the site ofrepair, it has been found that supraphysiological levels of BMP arerequired in order to promote healing due to the rapid diffusion ofgrowth factors out of the matrix. For example, with a collagen spongedelivery system, only 50% of the BMP added to the sponge is retainedafter two days (Geiger et al. (2003) Adv. Drug Del. Rev. 55: 1613-1629).The high initial dose of BMPs required to maintain physiological levelsof BMP for the necessary period of time makes BMP treatment moreexpensive and may lead to detrimental side effects such as ectopic boneformation or allergic reactions, or the formation of neutralizingantibodies.

Similar problems exist with other implants such as tendon and ligamentreplacements, skin replacements, vascular prostheses, heart pacemakers,artificial heart valves, breast implants, penile implants, stents,catheters, shunts, nerve growth guides, intraocular lenses, wounddressings, and tissue sealants. As with orthopedic implants, surgeryinvolving these implants often gives rise to similar problems with theslow healing of wounds and, where desirable, improper integration of theimplant into surrounding tissue.

Thus, there remains a need for the development of cost-effective methodsfor grafting active biomolecules to the surface of materials used asimplants or in conjunction with implants in order to promotepost-surgical healing and, where desirable, integration of the implantinto surrounding tissues, such as, for example, adjacent bone.

SUMMARY OF THE INVENTION

The present invention provides an improved coating for surfaces ofmedical implants. The coating comprises at least one interfacialbiomaterial (IFBM) which is comprised of at least one binding modulethat binds to the surface of an implant or implant-related material(“implant module”) and at least one binding module that binds to atarget analyte or that is designed to have a desired effect (“analytemodule”). The modules are connected by a linker. In some embodiments,the IFBM coating acts to promote the recognition and attachment oftarget analytes to surface of the device. The IFBM coating improves theperformance of implanted medical devices by promoting osteointegrationof the implant, accelerating healing, and/or reducing inflammation atthe site of the implant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of the binding of phage that displayrepresentative titanium-binding peptides to titanium beads (see Example1). Signal of assay for binding to titanium beads (vertical axis) isshown for various phage (horizontal axis).

FIG. 2 shows a comparison of the binding of peptides with a C-terminalbiotin residue to titanium (see Example 1). Absorbance (vertical axis)is shown as a function of peptide concentration (μM, on the horizontalaxis).

FIG. 3 shows a comparison of binding to titanium of two peptides (seeExample 2). A405 nm signal (vertical axis) is shown as a function ofpeptide concentration (μM, on the horizontal axis). The lines shown onthe graph from top to bottom join data points for peptides AFF6007 andAFF6010, respectively.

FIG. 4 shows a comparison of binding of various peptides to BMP-2 (seeExample 3). Signal (rate AP) is shown for various peptides (identifiedon the horizontal axis).

FIG. 5 shows the effect of BMP on the binding of IFBMs to a collagensponge (see Example 4). Signal (vertical axis) is shown as a function ofBMP concentration in nM (horizontal axis).

FIGS. 6A, 6B, 6C, and 6D show the results of an experiment described inExample 4 which demonstrates that the binding of BMP to collagen via anIFBM is dependent on both the amount of BMP put into the sponge and alsoon the amount of IFBM present. Absorbance (vertical axis) is shown as afunction of BMP concentration (horizontal axis).

FIG. 7 shows the results of an analysis of all the peptide sequencesfrom Tables 3 and 4 that bind BMP-2 and contain Motif 1 (see Example 3).The figure shows for each analyzed position the number of times eachamino acid was found in that position in the peptide sequences analyzed;for example, “G2” in position 1 means that Glycine was found two timesin that position.

FIG. 8 shows the oligonucleotide cassette which was designed to expressa peptide (SEQ ID NO: 74) containing the core binding Motif 1a in thecontext of a peptide sequence which also contained consensus residuesidentified for other positions in the sequence (see Example 3). Thenucleotide sequences shown in the figure are also set forth in SEQ IDNO: 75 and SEQ ID NO: 76.

FIG. 9 shows results from a conventional ELISA performed to evaluate therelative affinity of BMP binding peptides (see Example 3). The signalfrom the ELISA (A405 nm reading) is presented on the vertical axis as afunction of microliters of phage on the horizontal axis. At the datapoints corresponding to 0.10 microliters of phage, the lines shown onthe graph from top to bottom join data points for: APO2-61, APO2-40,APO2-41, APO2-26, APO2-35, APO2-59, APO2-44, mAEK, and the no-phagecontrol, respectively.

FIG. 10 shows the results of an analysis of all the peptide sequencesfrom Tables 3 and 5 that bind BMP-2 and contain Motif 2. The figureshows for each analyzed position the number of times each amino acid wasfound in that position in the peptide sequences analyzed; for example,“G7” in position 1 means that Glycine was found seven times in thatposition. Also shown are a consensus sequence derived from an alignmentof the peptides from Tables 3 and 5 that contain Motif 2 (SEQ ID NO:93). This sequence represents the predominant amino acid found at eachposition after all the peptides are aligned. Among the sequencesexamined, the most conserved amino acids form a core binding motifdesignated “Motif 2a” (SEQ ID NO: 94).

FIG. 11 shows representative results from an alternate assay forBMP-binding activity in which binding occurs in the solution phase (seeExample 3). Absorbance at 405 nm (vertical axis) is shown as a functionof picomoles of BMP (horizontal axis). These results were used tocalculate the affinity of each BMP-binding peptide for BMP-2 (see Table6). At the data point corresponding to one picomole of BMP, the linesshown on the graph from top to bottom join data points for: 2006, 2007,2008, 2009, 2011, and 2012, respectively.

FIG. 12 shows results from an assay in which several peptides weretested for their ability to bind to BMP-2, BMP-4, and BMP-7 (see Example3). The 2007 and 2011 peptides were originally identified as BMP-2binding peptides, while the 9001 peptide was originally identified asbinding to an unrelated target.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an improved coating for surfaces ofmedical devices to promote the attachment of peptides, proteins, drugs,or cells to the device. The coating is an interfacial biomaterial (IFBM)that comprises multiple binding modules that are linked. The IFBMcomprises at least one binding module which binds to the surface of theimplant (“implant module”) and at least one binding module that binds toa target analyte or has a desired effect (“analyte module”). Exemplarybinding modules comprise the peptide sequences provided, for example, inthe sequence listing (SEQ ID NOs: 1-74 and 77-558). The modules areconnected by a linker In some embodiments, the binding of the bindingmodule of an IFBM to the surface of an implant is non-covalent.Similarly, in some embodiments, the binding of an analyte module to atarget analyte is non-covalent. According to one embodiment, the implantmodule and the analyte module comprise two separate peptide moleculessuch that the implant module binds to an implant material and theanalyte module binds specifically to a growth factor or cell. In someembodiments, the implant module and the analyte module are linked by acentral macromolecule. These binding modules typically bindnon-covalently to the implant material or target analyte, respectively.In embodiments where the analyte module does not bind to a targetanalyte but rather has a desired effect, the analyte module may, forexample, simulate the action of a growth factor by acting to recruitcells to the location of the implant. The IFBM selection method andstructure are described in U.S. patent application Ser. No. 10/300,694,filed Nov. 20, 2002 and published on Oct. 2, 2003 as publication number20030185870, which is herein incorporated by reference.

By “binds specifically” or “specific binding” is intended that theimplant module or analyte module binds to a selected implant material orto a selected analyte. In some embodiments, a module that bindsspecifically to a particular implant material or analyte binds to thatmaterial or analyte at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 200%, 300%, 400%, 500%, or a higher percentage more than themodule binds to an appropriate control such as, for example, a differentmaterial that is used in implants, a material that is not used inimplants, or a protein typically used for such purposes such as bovineserum albumin. By “analyte” is intended any substance or moiety thatimproves osteointegration of an implant or promotes or accelerateshealing of the surrounding tissues following implant surgery. Suitableanalytes which are binding targets for analyte modules include, but arenot limited to, growth factors such as bone morphogenic proteins (BMPs,such as, for example, BMP-7 and BMP-2), vascular endothelial growthfactor (VEGF), platelet-derived growth factor (PDGF), transforminggrowth factor-β (TGF-β), insulin growth factor-1 (IGF-1), insulin growthfactor-2 (IGF-2), fibroblast growth factor (FGF), nerve growth factor(NGF), and placental growth factor. Suitable analytes also includehormones, enzymes, cytokines, and other bioactive substances or moietieswhich are useful in obtaining the goals of the invention; that is, topromote osteointegration of an implant and/or to improve healing ofsurrounding tissues following implant surgery. Suitable analytes alsoinclude cells, for example, osteoblasts, chondrocytes, stem cells,progenitor cells, platelets, and other cells which perform roles inosteointegration and healing. In some embodiments, analyte modules cancomprise peptide sequences that bind cells or have bioactivity throughbinding to cells or receptors such as, for example, the peptidesequences RGD, YIGSR, and IKVAV, which are known in the art to haveparticular biological activities. See, e.g., Hersel et al. (2003)Biomaterials 24: 4385-4415; Grant et al. (1990) Ann. N.Y. Acad. Sci.588: 61-72; Hosokawa et al. (1999) Dev. Growth Differ. 41: 207-216. Insome embodiments, analyte modules comprise peptide sequences which bindto and/or mimic the effect of BMP-2, such as the exemplary sequences setforth in SEQ ID NOs: 11-28, 44-74, or 77-94. An analyte module thatbinds to cells can comprise a peptide that comprises a general cellattachment sequence that binds to many different cell types, or it cancomprise a peptide that binds to a specific cell type such as anosteoblast, a chondrocyte, an osteoprogenitor cell, or a stem cell.

The term “implant” generally refers to a structure that is introducedinto a human or animal body to restore a function of a damaged tissue orto provide a new function. An implant device can be created using anybiocompatible material to which binding agents can specifically bind asdisclosed herein. Representative implants include but are not limitedto: hip endoprostheses, artificial joints, jaw or facial implants,tendon and ligament replacements, skin replacements, bone replacementsand artificial bone screws, bone graft devices, vascular prostheses,heart pacemakers, artificial heart valves, breast implants, penileimplants, stents, catheters, shunts, nerve growth guides, intraocularlenses, wound dressings, and tissue sealants. Implants are made of avariety of materials that are known in the art and include but are notlimited to: a polymer or a mixture of polymers including, for example,polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acidcopolymers, polyanhidrides, polyorthoesters, polystyrene, polycarbonate,nylon, PVC, collagen (including, for example, processed collagen such ascross-linked collagen), glycosaminoglycans, hyaluronic acid, alginate,silk, fibrin, cellulose, and rubber; plastics such as polyethylene(including, for example, high-density polyethylene (HDPE)), PEEK(polyetheretherketone), and polytetrafluoroethylene; metals such astitanium, titanium alloy, stainless steel, and cobalt chromium alloy;metal oxides; non-metal oxides; silicone; bioactive glass; ceramicmaterial such as, for example, aluminum oxide, zirconium oxide, andcalcium phosphate; other suitable materials such as demineralized bonematrix; and combinations thereof. The term “polymer” as used hereinrefers to any of numerous natural and synthetic compounds of usuallyhigh molecular weight consisting of up to millions of repeated linkedunits, each a relatively simple molecule. The term “implant” as usedherein includes implant-related materials that are associated with theimplant and are also introduced into a human or animal body inconjunction with the implant.

In one embodiment of the invention, an IFBM creates a binding interfacethat mediates the attachment of growth factors to the surface of animplant. In some embodiments, implants prepared according to the methodsof the invention will have growth factors specifically attached to thesurface of the implant; the rate of diffusion of the growth factor awayfrom the site of the implant can vary depending on the affinity of theanalyte module for the growth factor in question and thus implants canbe prepared with varying rates of diffusion of growth factors. Inembodiments involving the attachment of growth factors to the surface ofan implant, the growth factor will have a positive effect such as, forexample, accelerating the healing process, reducing the amount of growthfactor required for healing, and minimizing the side effects caused byusing supraphysiological doses of the growth factor. Growth factors ofparticular interest either as analyte modules or as factors that bind toanalyte modules include, for example, BMP-2, BMP-7, PDGF, FGF, and TGFβ.

Thus, the present invention provides methods for preparing an implant tobe surgically placed into a patient wherein the device is coated with alayer comprising at least one IFBM. In some embodiments, the methodcomprises the steps of: (a) applying an IFBM coating to the implant,wherein the IFBM comprises an implant module that specifically binds tothe implant and an analyte module that specifically binds a growthfactor; (b) applying the growth factor to the surface of the implant bydipping, spraying, or brushing a solution containing the growth factoronto the implant; (c) placing the implant into a subject usingappropriate surgical techniques which will be known to those of skill inthe art.

Alternatively, a method for coating an implant so that the implanteddevice promotes growth factor attachment comprises the steps of: (a)applying an IFBM coating to the implant, wherein the IFBM comprises animplant module that specifically binds the implant and an analyte modulethat specifically binds growth factor at an implant site; and (b)placing the implant in a subject at the implant site; whereby growthfactor produced in the host binds to the implant via the IFBM. Theenhanced presence of growth factor at the implant site enhances healingof adjacent tissue and integration of the implant into the adjacenttissue.

In one embodiment of the invention, an IFBM mediates cell attachment tothe surface of an implant. By enhancing cell adhesion and tissueintegration, the IFBMs of the invention can accelerate healing andimprove the function of the implanted device. Thus, in accordance withthe present invention, a method for preparing an implant to besurgically placed into a patient can comprise: (a) applying an IFBMcoating to the implant, wherein the IFBM comprises at least one implantmodule that specifically binds the implant and at least one analytemodule that specifically binds to at least one type of cell; and (b)placing the implant in a subject at the implant site, whereby cells bindto the IFBM coating on the implant.

In some embodiments, a method for preparing an implant comprises: (a)applying an IFBM coating to the implant, wherein the IFBM comprises atleast one implant module that specifically binds the implant and atleast one analyte module that specifically binds at least one type ofcell; and (b) applying cells to the surface of the implant, for example,by dipping the implant into a solution containing the cells or brushinga solution containing the cells onto the implant. The implant may thenbe placed into a subject (i.e., a human patient or an animal patient).By “patient” as used herein is intended either a human or an animalpatient.

In another embodiment of the invention, an implant is coated with morethan one type of IFBM in order to provide a coating with multiplefunctionalities. For example, an implant coating can comprise a firstIFBM having an analyte module that binds a cell and a second IFBM havingan analyte module that binds a growth factor. A coating comprising theseIFBMs would bind both cells and growth factor to the surface of theimplant. In some embodiments, these IFBMs would be intermingled in thecoating so that the bound growth factor is in close proximity to thebound cells. In one embodiment, a coating comprises an IFBM that bindsto mesenchymal stem cells and an IFBM that binds to the growth factorBMP-2; the BMP-2 would trigger the differentiation of the stem cellsinto osteoblasts. In other embodiments, an implant coating can comprisea mixture of at least two different IFBMs which differ in either or boththeir implant module and their analyte module. In another embodiment, acoating comprises a multi-functional IFBM which has two analyte modules,one of which binds to a cell and one of which binds to a growth factor.

Binding modules (i.e., implant modules and/or analyte modules) may bepeptides, antibodies or antibody fragments, polynucleotides,oligonucleotides, complexes comprising any of these, or variousmolecules and/or compounds. Binding modules which are peptides may beidentified as described in pending U.S. patent application Ser. No.10/300,694, filed Nov. 20, 2002 and published on Oct. 2, 2003 aspublication number 20030185870. In some embodiments, binding modules maybe identified by screening phage display libraries for binding tomaterials including biocompatible materials (i.e., “biomaterials”) suchas titanium, stainless steel, cobalt-chrome alloy, polyurethane,polyethylene or silicone.

In some embodiments of the invention, the analyte module is a bioactivepeptide or binds to a bioactive peptide. These bioactive peptides may befragments of native proteins that retain the biological effect of thenative protein, as is well-known in the art. For example, TP508 is asynthetic peptide derived from thrombin which represents amino acids183-200 of human thrombin and has been shown to accelerate fracturehealing (see, e.g., Wang et al. (2002) Trans ORS 27: 234). TP508function is believed to be mediated by an RGD sequence within thepeptide that binds to integrins present on the cell surface (see, e.g.,Tsopanoglou et al. (2004) Thromb Haemost. 92(4):846-57.) Similarly, P-15is a 15 amino acid peptide derived from Type I collagen that representsthe cell-binding domain of collagen (see, e.g., Yang et al. (2004)Tissue Eng. 10(7-8): 1148-59). P-15 has been shown to enhance new boneformation (see, e.g., Scarano et al. (2003). Implant Dent. 12(4):318-24.). Bioactive peptides can also be fragments of growth factors.For example, Saito et al. (J Biomed Mater Res A. 2005 72A(1): 77-82)have shown that a synthetic peptide representing amino acids 73-92 ofBMP-2 retains BMP-2 biological activities including binding to a BMP-2receptor, activating gene expression and inducing ectopic boneformation.

Any implant module may be combined with any analyte module to create anIFBM of the invention so long as the desired activity is provided; thatis, so long as the IFBM specifically binds to a suitable implant and hasa suitable effect conferred by the analyte module, i.e., the ability tobind to BMP-2. One of skill in the art will appreciate that a variety oftypes and numbers of implant modules may be combined with a variety oftypes and numbers of analyte modules to create an IFBM of the invention.Thus, for example, one or more implant modules may be linked with one ormore analyte modules to create an IFBM. One of skill will be able toselect suitable implant module(s) and analyte module(s) depending on thematerial of which an implant is made and the desired activity to beconferred by the analyte module(s).

The term “antibody” as used herein includes single chain antibodies.Thus, an antibody useful as a binding module may be a single chainvariable fragment antibody (scFv). A single chain antibody is anantibody comprising a variable heavy and a variable light chain that arejoined together, either directly or via a peptide linker, to form acontinuous polypeptide. The term “single chain antibody” as used hereinencompasses an immunoglobulin protein or a functional portion thereof,including but not limited to a monoclonal antibody, a chimeric antibody,a hybrid antibody, a mutagenized antibody, a humanized antibody, andantibody fragments that comprise an antigen binding site (e.g., F_(ab)and F_(v) antibody fragments).

Phage display technology is well-known in the art. Using phage display,a library of diverse peptides can be presented to a target substrate,and peptides that specifically bind to the substrate can be selected foruse as binding modules. Multiple serial rounds of selection, called“panning,” may be used. As is known in the art, any one of a variety oflibraries and panning methods can be employed to identify a bindingmodule that is useful in the methods of the invention. For example,libraries of antibodies or antibody fragments may be used to identifyantibodies or fragments that bind to particular cell populations or toviruses (see, e.g., U.S. Pat. Nos. 6,174,708; 6,057,098; 5,922,254;5,840,479; 5,780,225; 5,702,892; and 5,667,988). Panning methods caninclude, for example, solution phase screening, solid phase screening,or cell-based screening. Once a candidate binding module is identified,directed or random mutagenesis of the sequence may be used to optimizethe binding properties of the binding module. The terms “bacteriophage”and “phage” are synonymous and are used herein interchangeably.

A library can comprise a random collection of molecules. Alternatively,a library can comprise a collection of molecules having a bias for aparticular sequence, structure, or conformation. See, e.g., U.S. Pat.Nos. 5,264,563 and 5,824,483. Methods for preparing libraries containingdiverse populations of various types of molecules are known in the art,and numerous libraries are also commercially available. Methods forpreparing phage libraries can be found, for example, in Kay et al.(1996) Phage Display of Peptides and Proteins (San Diego, AcademicPress); Barbas (2001) Phage Display: A Laboratory Manual (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.)

A binding module (i.e., implant module or analyte module) that is apeptide comprises about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 200, or up to 300 aminoacids. Peptides useful as a binding module can be linear, branched, orcyclic, and can include non-peptidyl moieties. The term “peptide”broadly refers to an amino acid chain that includes naturally occurringamino acids, synthetic amino acids, genetically encoded amino acids,non-genetically encoded amino acids, and combinations thereof. Peptidescan include both L-form and D-form amino acids.

A peptide useful as a binding module can be subject to various changes,substitutions, insertions, and deletions where such changes provide forcertain advantages in its use. Thus, the term “peptide” encompasses anyof a variety of forms of peptide derivatives including, for example,amides, conjugates with proteins, cyclone peptides, polymerizedpeptides, conservatively substituted variants, analogs, fragments,chemically modified peptides, and peptide mimetics. Any peptide that hasdesired binding characteristics can be used in the practice of thepresent invention.

Representative non-genetically encoded amino acids include but are notlimited to 2-aminoadipic acid; 3-aminoadipic acid; β-aminopropionicacid; 2-aminobutyric acid; 4-aminobutyric acid (piperidinic acid);6-aminocaproic acid; 2-aminoheptanoic acid; 2-aminoisobutyric acid;3-aminoisobutyric acid; 2-aminopimelic acid; 2,4-diaminobutyric acid;desmosine; 2,2′-diaminopimelic acid; 2,3-diaminopropionic acid;N-ethylglycine; N-ethylasparagine; hydroxylysine; allo-hydroxylysine;3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine;N-methylglycine (sarcosine); N-methylisoleucine; N-methylvaline;norvaline; norleucine; and ornithine.

Representative derivatized amino acids include, for example, thosemolecules in which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups can be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups canbe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine can be derivatized to form N-im-benzylhistidine.

The term “conservatively substituted variant” refers to a peptide havingan amino acid residue sequence substantially identical to a sequence ofa reference peptide in which one or more residues have beenconservatively substituted with a functionally similar residue such thatthe “conservatively substituted variant” will bind to the same bindingpartner with substantially the same affinity as the parental variant andwill prevent binding of the parental variant. In one embodiment, aconservatively substituted variant displays a similar bindingspecificity when compared to the reference peptide. The phrase“conservatively substituted variant” also includes peptides wherein aresidue is replaced with a chemically derivatized residue.

Examples of conservative substitutions include the substitution of onenon-polar (hydrophobic) residue such as isoleucine, valine, leucine ormethionine for another; the substitution of one aromatic residue such astryptophan, tyrosine, or phenylalanine for another; the substitution ofone polar (hydrophilic) residue for another such as between arginine andlysine, between glutamine and asparagine, between glycine, alanine,threonine and serine; the substitution of one basic residue such aslysine, arginine or histidine for another; or the substitution of oneacidic residue such as aspartic acid or glutamic acid for another.

While exemplary peptide sequences for use as binding modules in IFBMs ofthe invention are disclosed herein (e.g., in the sequence listing in SEQID NOs: 1-74 and 77-558), one of skill will appreciate that the bindingor other properties conferred by those sequences may be attributable toonly some of the amino acids comprised by the sequences. Peptides whichare binding modules of the present invention also include peptideshaving one or more substitutions, additions and/or deletions of residuesrelative to the sequence of an exemplary peptide sequence as disclosedherein, so long as the desired binding properties of the binding moduleare retained. Thus, binding modules of the invention include peptidesthat differ from the exemplary sequences disclosed herein by about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aminoacids, but that retain the ability of the corresponding exemplarysequence to bind to a particular material or to act as an analytemodule. A binding module of the invention that differs from an exemplarysequence disclosed herein will retain at least 25%, 50%, 75%, or 100% ofthe activity of a binding module comprising an entire exemplary sequencedisclosed herein as measured using an appropriate assay.

That is, binding modules of the invention include peptides that sharesequence identity with the exemplary sequences disclosed herein of atleast 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequenceidentity. Sequence identity may be calculated manually or it may becalculated using a computer implementation of a mathematical algorithm,for example, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package of Genetics Computer Group, Version 10(available from Accelrys, 9685 Scranton Road, San Diego, Calif., 92121,USA). The scoring matrix used in Version 10 of the Wisconsin GeneticsSoftware Package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc.Nat'l. Acad. Sci. USA 89: 10915). Alignments using these programs can beperformed using the default parameters.

A peptide can be modified, for example, by terminal-NH₂ acylation (e.g.,acetylation, or thioglycolic acid amidation) or byterminal-carboxylamidation (e.g., with ammonia or methylamine). Terminalmodifications are useful to reduce susceptibility by proteinasedigestion, and to therefore prolong a half-life of peptides insolutions, particularly in biological fluids where proteases can bepresent.

Peptide cyclization is also a useful modification because of the stablestructures formed by cyclization and in view of the biologicalactivities observed for such cyclic peptides. Methods for cyclizingpeptides are described, for example, by Schneider & Eberle (1993)Peptides. 1992: Proceedings of the Twenty-Second European PeptideSymposium, Sep. 13-19, 1992, Interlaken, Switzerland, Escom, Leiden, TheNetherlands.

Optionally, a binding module peptide can comprise one or more aminoacids that have been modified to contain one or more halogens, such asfluorine, bromine, or iodine, to facilitate linking to a linkermolecule. As used herein, the term “peptide” also encompasses a peptidewherein one or more of the peptide bonds are replaced by pseudopeptidebonds including but not limited to a carba bond (CH₂—CH₂), a depsi bond(CO—O), a hydroxyethylene bond (CHOH—CH₂), a ketomethylene bond(CO—CH₂), a methylene-oxy bond (CH₂—O), a reduced bond (CH₂—NH), athiomethylene bond (CH₂—S), an N-modified bond (—NRCO—), and athiopeptide bond (CS—NH). See e.g., Garbay-Jaureguiberry et al. (1992)Int. J. Pept. Protein Res. 39: 523-527; Tung et al. (1992) Pept. Res. 5:115-118; Urge et al. (1992) Carbohydr. Res. 235: 83-93; Corringer et al.(1993) J. Med. Chem. 36: 166-172; Pavone et al. (1993) Int. J. Pept.Protein Res. 41: 15-20.

Representative peptides that specifically bind to surfaces of interest(including titanium, stainless steel, collagen, and poly glycolic acid(PGA)) and therefore are suitable for use as binding modules in IFBMs ofthe invention are set forth in the sequence listing and are furtherdescribed herein below. While exemplary peptide sequences are disclosedherein, one of skill will appreciate that the binding propertiesconferred by those sequences may be attributable to only some of theamino acids comprised by the sequences. Thus, a sequence which comprisesonly a portion of an exemplary sequence disclosed herein may havesubstantially the same binding properties as the full-length exemplarysequence. Thus, also useful as binding modules are sequences thatcomprise only 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the amino acids in aparticular exemplary sequence, and such amino acids may be contiguous ornon-contiguous in the exemplary sequence. Such amino acids may beconcentrated at the amino-terminal end of the exemplary peptide (forexample, 4 amino acids may be concentrated in the first 5, 6, 7, 8, 9,10, 11, or 12 amino acids of the peptide) or they may be dispersedthroughout the exemplary peptide but nevertheless be responsible for thebinding properties of the peptide. For example, a peptide thatspecifically binds to BMP-2 may comprise all or part of a sequence motifsuch as that described in Example 3 and set forth in SEQ ID NO:27 or 28.Thus, a peptide that specifically binds to BMP-2 may have a sequencethat conforms to each requirement of the sequence motif as set forth inSEQ ID NO:27 or 28, or it may have a sequence that conforms to 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 of the requirements of the sequence motif. Thesequence motif set forth in SEQ ID NO:27 can be described as having four“requirements” which limit the amino acids that are present at positions1, 4, 6, and 7. A peptide that specifically binds to BMP-2 may have asequence as set forth in SEQ ID NO:11 which conforms to all four ofthose requirements, or it may have a sequence as set forth in SEQ IDNO:21 which conforms to three of those four requirements. Both of thesetypes of sequences are provided by the present invention.

In some embodiments, the IFBM has been constructed so as to mimic thebiological effects of protein growth factors. In these embodiments, theanalyte module comprises a peptide which comprises an amino acidsequence which binds to the BMP receptor BMPRI and also comprises anamino acid sequence which binds to the BMP receptor BMPRII (see, forexample, Example 6). These receptors are well-known in the art and arealso commercially available (for example, from R&D Systems, Minneapolis,Minn., Cat. Nos. 315-BR and 811-BR). In these embodiments, the analytemodule has BMP activity as measured, for example, by techniques known inthe art and described in Example 6. While the invention is not bound byany particular mechanism of operation, it is believed that by binding toeach of BMPRI and BMPRII, the analyte module will encourage theheterodimerization of these receptors, thereby triggering signaling viathe BMP-SMAD pathway. In this manner, an IFBM could be constructed andused to coat the surface of an implant so as to trigger signaling viathe BMP-SMAD pathway without the addition of BMP itself. Generally, inthe native BMP-SMAD pathway, heterodimerization of the BMP type I andtype II receptors is required for signaling (see, e.g., Chen et al.(2004) Growth Factors 22: 233-241). Dimerization brings the cytoplasmicdomains of the type I and type II receptors into proximity, allowing theconstitutively active type II receptor kinase to phosphorylate the typeI receptor. The phosphorylation of the cytoplasmic domain of the type Ireceptor activates its latent kinase activity which in turn activatesSmad proteins. After release from the receptor, the phosphorylated Smadproteins associate with Smad4 and this complex is translocated into thenucleus to function with other proteins as transcription factors andregulate responsive genes (Chen et al. (2004) Growth Factors 22:233-241). Collectively, this can be referred to as the downstream Smador BMP-SMAD signal transduction pathway and genes activated thereby.Proteins produced as a result of activation of the Smad or BMP-SMADpathway can be referred to as Smad-activated downstream proteinproducts.

Binding modules of the present invention that are peptides can besynthesized by any of the techniques that are known to those skilled inthe art of peptide synthesis. Representative techniques can be found,for example, in Stewart & Young (1969) Solid Phase Peptide Synthesis,(Freeman, San Francisco, Calif.); Merrifield (1969) Adv. Enzymol. Relat.Areas Mol. Biol. 32: 221-296; Fields & Noble (1990) Int. J. Pept.Protein Res. 35: 161-214; and Bodanszky (1993) Principles of PeptideSynthesis, 2nd Rev. Ed. (Springer-Verlag, Berlin). Representative solidphase synthesis techniques can be found in Andersson et al. (2000)Biopolymers 55: 227-250, references cited therein, and in U.S. Pat. Nos.6,015,561; 6,015,881; 6,031,071; and 4,244,946. Peptide synthesis insolution is described in Schroder & Lake (1965) The Peptides (AcademicPress, New York, N.Y.). Appropriate protective groups useful for peptidesynthesis are described in the above texts and in McOmie (1973)Protective Groups in Organic Chemistry (Plenum Press, London). Peptides,including peptides comprising non-genetically encoded amino acids, canalso be produced in a cell-free translation system, such as the systemdescribed by Shimizu et al. (2001) Nat Biotechnol 19: 751-755. Inaddition, peptides having a specified amino acid sequence can bepurchased from commercial sources (e.g., Biopeptide Co., LLC of SanDiego, Calif.), and PeptidoGenics of Livermore, Calif.).

The binding modules are connected by at least one linker to form an IFBMof the invention. In some embodiments, IFBMs consisting of bindingmodules which are peptides are synthesized as a single continuouspeptide; in these embodiments, the linker is simply one of the bonds inthe peptide. In other embodiments of the invention, a linker cancomprise a polymer, including a synthetic polymer or a natural polymer.Representative synthetic polymers which are useful as linkers includebut are not limited to: polyethers (e.g., polyethylene glycol; PEG),polyesters (e.g., polylactic acid (PLA) and polyglycolic acid (PGA),polyamides (e.g., nylon), polyamines, polyacrylic acids, polyurethanes,polystyrenes, and other synthetic polymers having a molecular weight ofabout 200 daltons to about 1000 kilodaltons. Representative naturalpolymers which are useful as linkers include but are not limited to:hyaluronic acid, alginate, chondroitin sulfate, fibrinogen, fibronectin,albumin, collagen, and other natural polymers having a molecular weightof about 200 daltons to about 20,000 kilodaltons. Polymeric linkers cancomprise a diblock polymer, a multi-block copolymer, a comb polymer, astar polymer, a dendritic polymer, a hybrid linear-dendritic polymer, ora random copolymer.

A linker can also comprise a mercapto(amido)carboxylic acid, anacrylamidocarboxylic acid, an acrlyamido-amidotriethylene glycolic acid,and derivatives thereof. See, for example, U.S. Pat. No. 6,280,760.Where a linker comprises a peptide, the peptide can include sequencesknown to have particular biological functions, such as YGD and GSR.

Methods for linking a linker molecule to a binding domain will varyaccording to the reactive groups present on each molecule. Protocols forlinking using reactive groups and molecules are known to one of skill inthe art. See, e.g., Goldman et al. (1997) Cancer Res. 57: 1447-1451;Cheng (1996) Hum. Gene Therapy 7: 275-282; Neri et al. (1997) Nat.Biotechnol. 19: 958-961; Nabel (1997) Current Protocols in HumanGenetics, vol. on CD-ROM (John Wiley & Sons, New York); Park et al.(1997) Adv. Pharmacol. 40: 399-435; Pasqualini et al. (1997) Nat.Biotechnol. 15: 542-546; Bauminger & Wilchek (1980) Meth. Enzymol. 70:151-159; U.S. Pat. Nos. 6,280,760 and 6,071,890; and European PatentNos. 0 439 095 and 0 712 621.

The surfaces of medical devices are coated by any suitable method, forexample, by dipping, spraying, or brushing the IFBM onto the device. Thecoating may be stabilized, for example, by air drying or bylyophilization. However, these treatments are not exclusive, and othercoating and stabilization methods may be employed. Suitable methods areknown in the art. See, e.g., Harris et al. (2004) Biomaterials 25:4135-4148 and U.S. patent application Ser. No. 10/644,703, filed Aug.19, 2003 and published on May 6, 2004 with Publication No. 20040087505.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

EXPERIMENTAL Example 1 Isolation of Peptides that Bind Titanium

Ten different phage display libraries were screened for binding totitanium beads. Titanium (Ti₆Al₄V) beads of approximately 5/32 of aninch diameter were washed with 70% ethanol, 40% nitric acid, distilledwater, 70% ethanol, and acetone to remove any surface contaminants. Onetitanium bead was placed per well of 96-well polypropylene plate (Nunc).

Nonspecific binding sites on the titanium and the surface of thepolypropylene were blocked with 1% bovine serum albumin (BSA) inphosphate-buffered saline (PBS; Sigma Chemical Co., St. Louis, Mo., Cat.#P-3813). The plate was incubated for 1 hour at room temperature withshaking at 50 rpm. The wells were then washed 5 times with 300 μl ofPBS. Each library was diluted in PBS+1% BSA and was added at aconcentration of 10¹⁰ pfu/ml in a total volume of 250 μl. After a 3-hourincubation at room temperature and shaking at 50 rpm, unbound phage wereremoved by washing 3 time with 300 μl of Phosphate BufferedSaline-Tween™ 20 (PBS-T; Sigma Chemical Co., St. Louis, Mo., Cat.#P-3563). To recover the phage bound to the titanium beads, bound phagewere released by treating with 50 mM glycine, pH 2 for 10 minutesfollowed by a 10 minute treatment with 100 mM ethanolamine, pH 12. Theeluted phage were pooled, neutralized with 200 μl of 200 mM NaPO₄ pH 7.The eluted phage and the beads were added directly to E. coli DH5αF′cells in 2×YT media. The mixture was incubated overnight in a 37′Cshaker at 210 rpm. Phage supernatant was then harvested after spinningat 8500×g for 10 minutes. Second and third rounds of selection wereperformed in a similar manner to that of the first round, using the 50μl of amplified phage from the previous round as input diluted with 200μl of PBS+1% BSA. The fourth round of selection was carried out in asimilar fashion; however, the washes were modified. After a 4 hourbinding reaction, the beads were washed five times with PBS-T (SigmaChemical Co., St. Louis, Mo., Cat. #P-3563), the beads were moved to aclean polypropylene plate with 2 ml wells, 1 ml of PBS+1% BSA was addedto each well and the washing was incubated overnight at room temperaturewith shaking at 50 rpm. The next morning the phage were eluted andamplified in the same manner described for rounds 1-3. Individual clonalphage were then isolated and tested by plating out dilutions of phagepools to obtain single plaques.

To detect phage that specifically bound to titanium, conventional ELISAswere performed using an anti-M13 phage antibody conjugated to HRP,followed by the addition of chromogenic agent ABTS. Relative bindingstrengths of the phage were determined by testing serial dilutions ofthe phage for binding to titanium in an ELISA.

The DNA sequence encoding peptides that specifically bound titanium wasdetermined. The sequence encoding the peptide insert was located in thephage genome and translated to yield the corresponding amino acidsequence displayed on the phage surface.

Representative peptides that specifically bind titanium are listed inTable 1 and are set forth as SEQ ID NOs:1-8. The binding of phagedisplaying these peptides to titanium beads is shown in FIG. 1.

TABLE 1 Titanium Binding Peptides Synthetic SEQ. Clone Peptide ID.Number Number Displayed Peptide NO. AP06-22 AFF-6002 SSHKHPVTPRFFVVESR 1AP06-23 AFF-6003 SSCNCYVTPNLLKHKCYKICSR 2 AP06-24 AFF-6004SSCSHNHHKLTAKHQVAHKCSR 3 AP06-25 AFF-6005 SSCDQNDIFYTSKKSHKSHCSR 4AP06-26 AFF-6006 SSSSDVYLVSHKHHLTRHNSSR 5 AP06-27 AFF-6007SSSDKCHKHWYCYESKYGGSSR 6 AP06-28 HHKLKHQMLHLNGG 7 AP06-29 GHHHKKDQLPQLGG8

The displayed peptides were then synthesized with a C-terminal biotinresidue and tested for binding to titanium. Results are shown in FIG. 2.Briefly, peptide stock solutions were made by dissolving the powder in100% DMSO to make a 1 mM solution of peptide. Serial dilutions of thepeptide were made in PBS-T. Titanium beads blocked with 1% non-fat drymilk in PBS were incubated with various concentrations of peptide for 1hour at room temperature with shaking. The beads were washed 3 timeswith PBS-T. Streptavidin-alkaline phosphatase (SA-AP) from USB (UnitedStates Biochemical, catalog #11687) was added (1:1000 in PBS-T) andincubated 1 hour at room temperature with shaking. The beads were washed3 times with PBS-T and the amount of peptide:SA-AP was determined byadding PNPP (Sigma-Aldrich, Inc., SigmaFast tablets, catalog #N1891) andallowing the color to develop for about 10 minutes. Quantitation wascarried out by transferring the solution to a clear microtiter plate andreading the absorbance at 405 nm on a Molecular Dynamics Plate Reader.The peptide “9003” is known in the art. This peptide was identified byphage display as binding to the enzyme hexokinase; it serves as anegative control for this experiment (see, e.g., Hyde-DeRuyscher et al.(2000) Chem. Biol. 7: 17-25).

Example 2 Role of Cysteine Residues in Titanium-binding Peptide 6007

To explore the role of the cysteine residues and disulfide formation inthe binding of peptide 6007 to titanium, a peptide was synthesizedAFF6010 (Table 2) in which the cysteine residues present in thetitanium-binding peptide AFF6007 were changed to serine residues. Thesequence of peptide AFF6010 (SSSDKSHKHWYSYESKYGGSGSSGK) is set forth inSEQ ID NO:9, while the sequence of peptide AFF6007(SSSDKCHKHWYCYESKYGGSGSSGK) is set forth in SEQ ID NO:10. The peptidesAFF6007 and AFF6010 were then conjugated to biotin and compared forbinding to titanium beads as follows.

Titanium beads were blocked with 1% BSA in PBS for 30 minutes at roomtemperature. Stock solutions of peptide AFF6007 and AFF6010 wereprepared by dissolving 1-2 mg peptide in water. The final concentrationof each peptide was determined using the optical density at 280 nm andthe extinction coefficient of each peptide. AFF6007 and AFF6010 wereprepared at 200 μM. A dilution series was then prepared for each peptidesample. Each peptide underwent a threefold dilution in 1% BSA in PBS.

The peptides were incubated with the titanium beads for 1 hour at roomtemperature. Beads were then washed two times with PBS/Tween™ 20.Streptavidin-alkaline phosphatase was then added to the beads at 1:500for 30 minutes at room temperature. Beads were washed two times withPBS/Tween™ 20. PNPP was used to develop the assay and the absorbance wasrecorded at 405 nm.

The results, which are shown in FIG. 3, demonstrate that peptidesAFF6007 and AFF6010 both bind to titanium. An estimate of the relativeaffinity of a peptide for titanium can be made by determining theconcentration of peptide that gives one-half the maximal signal (Table2). The complete elimination of the cysteine residues in AFF6007decreases the affinity of the peptide for titanium by about 10-fold butdoes not eliminate it (Table 2). Therefore, the cysteine residues arenot required for binding to titanium but do increase the affinity of thepeptide for titanium.

TABLE 2 Relative Affinity of Titanium-binding Peptides [peptide] Sample½ maximal signal AFF6007 0.35 μM AFF6010   3 μM

Example 3 Peptides that Specifically Bind to Bone Morphogenic Protein 2(“BMP-2”) Isolation and Analysis of Peptides

Ten different phage display libraries were screened for binding toBMP-2. BMP-2 (Medtronic) was biotinylated with NHS-biotin (Pierce) toproduce a labeled protein with an average of one biotin per proteinmolecule. This protein was immobilized on streptavidin (SA) coatedplates and used as target for phage display. As an alternative method todisplay the protein, BMP-2 was also linked to sepharose beads usingNHS-succinimide chemistry according to the instructions of themanufacturer (Amersham-Pharmacia, Ref. No. 18-1022-29, entitled“Coupling through the Primary Amine of a Ligand to NHS-activatedSepharose 4 Fast Flow,” pp. 105-108) and the beads were used as a solidphase to separate free from unbound phage. After 3 rounds of selection,individual clones from each format were tested for binding to BMP-2 onSA coated plates utilizing a conventional ELISA using an anti-M13 phageantibody conjugated to HRP, followed by the addition of chromogenicagent ABTS.

The DNA sequence encoding peptides that specifically bound to BMP-2 wasdetermined. The sequence encoding the peptide insert was located in thephage genome and translated to yield the corresponding amino acidsequence displayed on the phage surface. Representative peptides thatspecifically bind BMP-2 are listed in Table 3 and are set forth as SEQID NOs:11-26. In some embodiments, an exemplary binding module of theinvention comprises only that portion of the sequence shown in uppercaseletters.

TABLE 3 Peptides that Specifically Bind to BMP-2 Synthetic SEQ. ClonePeptide ID. Number Number Sequence NO. AP02-45 AFF-2011

11 AP02-46

12 AP02-47

13 AP02-48 AFF-2006

14 AP02-49

15 AP02-50 AFF-2007

16 AP02-51

17 AP02-52 AFF-2010

18 AP02-53

19 AP02-54 AFF-2008

20 AP02-55

21 AP02-56

22 AP02-57

23 AP02-58

24 AP02-59 AFF-2012

25 AP02-60 AFF-2009

26

The peptides that were identified fall into 2 different “sequenceclusters”. Each sequence cluster contains a common sequence motif. Forthe first sequence cluster of BMP-binding peptides, the common motif(designated “Motif 1” and set forth in SEQ ID NO:27) isAromatic-X-X-Phe-X-“Small”-Leu (Aromatic=Trp, Phe, or Tyr; X=any aminoacid; “Small”=Ser, Thr, Ala, or Gly). Motif 1 is at least partiallyfound in SEQ ID NOs:11-24 as shown in Table 3 above. The second sequencecluster motif (also set forth in SEQ ID NO:28) comprises the sequence(Leu or Val)-X-Phe-Pro-Leu-(Lys or Arg)-Gly. This motif, designatedMotif 2, is found in SEQ ID NOs:25 and 26 as shown in Table 3 above.Exemplary binding modules also comprise sequences which meet therequirements of this or other sequence motifs identified herein (i.e.,which contain a sequence which falls within these motifs).

Additional experiments were conducted to determine additionalcharacteristics of sequences that bind to BMP-2. Specifically, in orderto determine whether there were additional preferred amino acidssurrounding these motifs, further screening was conducted. Focusedlibraries were designed and cloned into the mAEK phage display vectorand the resultant phage were screened for binding to BMP-2, as furtherdiscussed below. The focused library for Motif 1 was designed to expresspeptides containing the following sequence:X-X-X-X-X-(W/L/C/Y/F/S)-X-X-(W/L/C/Y/F/S)—X-(A/G/N/S/T)-(L/F/I/M/V)-X-X-X-X-X,where X represents any of the 20 naturally occurring amino acids andpositions in parentheses are restricted to the amino acids listed withinthe parentheses. These peptides were encoded by oligonucleotidescomprising the sequence5′-GATCCTCGAGNNNKNNKNNKNNKNNKTNBNNKNNKTNBNNKRSYNTKNNKNNKNNKNNKNNKTCTAGAGCGCTACG 3′ (where “N” is any of the 4 nucleotides A,G, C, or T; “K” is G or T; “R” is A or G; “S” is C or G; “B” is C, G, orT; and “Y” is C or T). The focused library for Motif 2 was designed toexpress peptides containing the following sequence:X-X-X-(L/F/I/M/V)-X-(W/L/C/Y/F/S)-(P/S/T/A)-(L/F/I/M/V)-(I/M/T/N/K/S/R)-X-X-X-X-X-X-X-X.These peptides were encoded by oligonucleotides comprising the sequence5′-GATCCTCGANNNKNNKNNKNTKNNKTNBNCKNTKANKNNKNNKNNKNNKNNKNNKNNKNNKTCTAGAGCGCTACG 3′.

The following is provided as an exemplary library construction schemefor the Motif 1 focused library. As will be appreciated by one of skillin the art, a similar strategy can be used for other libraries. Toproduce the focused library for Motif 1, an oligonucleotide comprisingthe sequence above flanked by appropriate restriction enzyme sites wassynthesized. This oligonucleotide contained the sequence5′-GATCCTCGAGNNNKNNKNNKNNKNNKTNBNNKNNKTNBNNKRSYNTKNNKNNKNNKNNKNNKTCTAGAGCGCTACG-3′. In this sequence, the underlined sequencesCTCGAG and TCTAGA represent the XhoI and XbaI restriction enzyme sitesused to clone the library into the phage vector. A short primer isannealed to the oligonucleotide and the complementary strand synthesizedusing a DNA polymerase. The resulting double-stranded DNA molecule isdigested with XhoI and XbaI and cloned into the phage display vector.The ligated DNA is transformed into an appropriate bacterial host andamplified to generate the phage library.

The focused libraries for Motif 1 and Motif 2 were screened for bindingto BMP-2 using biotinylated BMP-2 immobilized on streptavidin-coatedplates as described above. After two rounds of selection on BMP-2, thelibraries had been enriched for phage displaying peptides that bind toBMP-2. The pools of enriched phage were plated onto a lawn of bacterialcells to isolate individual phage. Individual phage clones were testedfor binding to BMP-2 using an ELISA-type assay and an anti-M13 phageantibody conjugated to HRP (Amersham Biosciences #27-9421-01), followedby addition of the chromogenic reagent ABTS (Sigma Chemical Co., St.Louis, Mo., Cat. #A3219).

The DNA sequence encoding peptides that specifically bound to BMP-2 wasdetermined. The sequence encoding the peptide insert was located in thephage genome and translated to yield the corresponding amino acidsequence displayed on the phage surface.

Representative peptides from the motif-based focused libraries thatspecifically bind BMP-2 are listed in Tables 4 and 5 and are set forthas SEQ ID NOs:44-71 and 77-92. In some embodiments, an exemplary bindingmodule of the invention comprises only that portion of the sequenceshown in uppercase letters, or comprises only a sequence falling withina motif or a consensus sequence identified based on these sequences(i.e., comprises a sequence falling within the scope of Motif 1, Motif1a, or Motif 2, or comprises the consensus sequence identified in SEQ IDNO:72, 74, or 93).

TABLE 4 BMP Binding Peptides from Motif 1 Focused Library SEQ. IDClone ID Sequence NO. AP02-01

44 AP02-02

45 AP02-03

46 AP02-04

47 AP02-05

48 AP02-06

49 AP02-07

50 AP02-08

51 AP02-09

52 AP02-10

53 AP02-11

54 AP02-12

55 AP02-13

56 AP02-14

57 AP02-15

58 AP02-16

59 AP02-17

60 AP02-18

61 AP02-19

62 AP02-20

63 AP02-21

64 AP02-22

65 AP02-23

66 AP02-24

67 AP02-25

68 AP02-26

69 AP02-27

70 AP02-44

71

The results of an analysis of all the peptide sequences from Tables 3and 4 that bind BMP-2 and contain Motif 1 was generated and is shown inFIG. 7. From an alignment of the 40 BMP-binding sequences that containMotif 1, a consensus sequence can be derived(Gly-Gly-Gly-Ala-Trp-Glu-Ala-Phe-Ser-Ser-Leu-Ser-Gly-Ser-Arg-Val; SEQ IDNO: 72) that represents the predominant amino acid found at eachposition after all the peptides are aligned. Among the 40 sequences, themost conserved amino acids form a core binding motif which represents asubset of all sequences containing Motif 1. This motif, designated“Motif 1a,” has the sequence Trp-X-X-Phe-X-X-Leu (SEQ ID NO: 73). Whilethe invention is not bound by any particular mechanism of action, it isbelieved that in this motif, the Trp, Phe, and Leu residues on thepeptide participate in specific interactions with the BMP-2 protein thatare responsible for the binding of the peptide to BMP. On this basis, itwas hypothesized that other peptides that contain this core bindingmotif will also bind to BMP.

To test this idea, an oligonucleotide cassette was designed to express apeptide which contained this core binding Motif 1a in the context of apeptide sequence which also contained consensus residues identified forother positions in the sequence that flanked the core binding motif (seeFIG. 8; SEQ ID NO: 74). Incidentally, none of the BMP-binding peptidespreviously isolated by phage display actually contain this exactsequence (see, e.g., Table 4). This oligonucleotide cassette was clonedinto the mAEK phage display vector and the resulting phage, designatedAP02-61, was tested for binding to BMP-2 and compared to other phagedisplaying BMP-binding peptides (results for some phage are shown inFIG. 9). At least one phage tested (designated AP02-37) showed bindingat a level equivalent to or below that of the display vector mAEK. Insome embodiments, an exemplary binding module of the invention comprisesonly that portion of the sequence shown in uppercase letters.

TABLE 5 BMP-Binding Peptides from Motif-2 Focused Library SEQ.  ID.Clone ID Sequence NO.: AP02-28

77 AP02-29

78 AP02-30

79 AP02-31

80 AP02-32

81 AP02-33

82 AP02-34

83 AP02-35

84 AP02-36

85 AP02-37

86 AP02-38

87 AP02-39

88 AP02-40

89 AP02-41

90 AP02-42

91 AP02-43

92

From an alignment of the peptides from Tables 3 and 5 that contain Motif2, a consensus sequence can be derived(Gly-Gly-Ala-Leu-Gly-Phe-Pro-Leu-Lys-Gly-Glu-Val-Val-Glu-Gly-Trp-Ala;SEQ ID NO: 93; see FIG. 10) that represents the predominant amino acidfound at each position after all the peptides are aligned. Among thesequences examined, the most conserved amino acids form a core bindingmotif designated “Motif 2a,” which has the sequenceLeu-X-Phe-Pro-Leu-Lys-Gly (SEQ ID NO: 94).

Motif 2 appears to be more restricted in sequence than Motif 1 in thatMotif 2 imposes requirements on six positions whereas Motif 1 onlyimposes requirements on three positions. The Pro and Gly residues inMotif 2 appear to be required for binding since every Motif 2-containingBMP-binding peptide contains the Pro and Gly residues found in the corebinding motif. Using the consensus sequence information for Motif 2,BMP-binding peptides can be designed by incorporating the Motif 2 corebinding motif into the peptide sequence.

Production of Synthetic Peptides and BMP-2 Binding Assays

A representative set of the displayed peptides were then synthesizedwith a C-terminal biotin residue and tested for binding to BMP-2.Results are shown in FIG. 4. Briefly, peptide stock solutions were madeby dissolving the powder in 100% DMSO to make a 10 mM solution ofpeptide, water was then added for a final stock concentration of peptideof 1 mM in 10% DMSO. Serial dilutions of the peptide were made in PBS-T.A dilution series of BMP-2 with concentrations ranging from 100 nM to0.1 nM was immobilized onto the wells of microtiter plates (Immulon-4®HBX from Dynex Technologies, Chantilly, Va.) and blocked with 1% BSA.These plates were incubated with various concentrations of peptide for 1hour at room temperature with shaking. The beads were washed 3 timeswith PBS-T. Streptavidin-alkaline phosphatase (SA-AP) from USB (UnitedStates Biochemical, catalog #11687) was added (1:1000 in PBS-T) andincubated 1 hour at room temperature with shaking. The plates werewashed 3 times with PBS-T and the amount of peptide:SA-AP was determinedby adding PNPP (Sigma-Aldrich, Inc., SigmaFast tablets, catalog #N1891)and allowing the color to develop for about 10 minutes. Quantitation wascarried out by reading the absorbance at 405 nm on a Molecular DynamicsPlate Reader. The results are summarized in FIG. 4.

To confirm these BMP binding results, the peptides were also tested inan alternate assay format in which the peptide and BMP2 were allowed tobind in solution and then assayed. Briefly, the peptides weresynthesized with a biotin group attached to the 8 amino group of alysine residue at the C-terminus of the peptide. The biotinylatedpeptides (0-12 pmoles) were mixed with BMP-2 (0-25 pmoles) in solutionand allowed to incubate at 37° C. for 30 minutes in a polypropyleneplate. The solutions were transferred to a streptavidin-coated plate andincubated for 1 hour at 37° C. to capture the biotinylated peptides.Plates were washed in TBS-Tween™ 20 and then incubated with an anti-BMPantibody (1:1000 dilution; R&D systems) for 1 hour at RT. After washing,an alkaline phosphatase-labeled secondary antibody was then added to theplate and incubated at RT for 30 minutes. The plates were washed withTBS-Tween™ 20 and the antibody binding was detected using thechromogenic AP substrate pNPP. Representative results are shown in FIG.11. From this data, the affinity of each BMP-binding peptide for BMP-2was calculated (Table 6).

TABLE 6 Estimated Affinity of BMP-binding Peptides for BMP-2 EstimatedPeptide Affinity (nM) 2012 9 2009 10 2006 21 2011 55 2007 79 2008 99BMP-2 Binding Peptides Bind to other BMP proteins

Bone Morphogenetic Proteins (BMPs) are members of the TGF-betasuperfamily which includes BMPs, Transforming Growth Factor-beta (TGF-β)and Growth/Differentiation factors (GDFs). The proteins in the TGF-βsuperfamily are very similar structurally. The folded structure of theprotein backbone is almost identical among all the members of thefamily. Based on the similarity in structure between the BMPs, we testedthe ability of some of the BMP-2-binding peptides to bind BMP-4 andBMP-7. Biotinylated peptides 2007 and 2011 were tested for binding toBMP-2, BMP-4, and BMP-7 as described above. Both 2007 and 2011 bound toall three BMPs while a peptide that binds to an unrelated target(AFF-9001) did not bind to any of the BMPs (FIG. 12).

Example 4 Generation of an IFBM that Immobilizes BMP-2 onto Collagen

To design a molecule with collagen and BMP-2 binding properties, an IFBMwas created that comprised a peptide that binds to collagen and apeptide that binds to BMP-2. Examples of this “hybrid peptide” IFBM areshown in Table 7.

TABLE 7 IFBMs that bind to Collagen and BMP-2 IFBM SEQ ID  # PeptidesPeptide Sequence NO: AFF 2009-SSFEPLRFPLKGVPVSRGSSGKDVNSIWMSRVIEWTYDS-NH2 29 7005 0016 AFF 0016-DVNSIWMSRVIEWTYDSGSSGKSSFEPLRFPLKGVPVSR-NH2 30 7006 2009 AFF 2006-SRSSDSAFSSFSALEGSVVSRGSSGKDVNSIWMSRVIEWTYDS-NH2 31 7007 0016 AFF 0016-DVNSIWMSRVIEWTYDSGSSGKSRSSDSAFSSFSALEGSVVSR-NH2 32 7008 2006 AFF 2012-SSSVDLYFPLKGDVVSRGSSGKDVNSIWMSRVIEWTYDS-NH2 33 7009 0016 AFF 0016-DVNSIWMSRVIEWTYDSGSSGKSSSVDLYFPLKGDVVSR-NH2 34 7010 2012 AFF 2007-SRGGEAAAGAWVSFSALESSRGSSGKDVNSIWMSRVIEWTYDS-NH2 35 7014 0016 AFF 0016-DVNSIWMSRVIEWTYDSGSSGKSRGGEAAAGAWVSFSALESSR-NH2 36 7015 2007 AFF 2011-SSDWGVVASAWDAFEALDASRGSSGKDVNSIWMSRVIEWTYDS-NH2 37 7016 0016 AFF 0016-DVNSIWMSRVIEWTYDSGSSGKSSDWGVVASAWDAFEALDASR-NH2 38 7017 2011

As shown in Table 7, each IFBM contains the collagen binding domain fromAFF0016 followed by a short linker sequence which is then linked to aBMP binding sequence from the above example in a “hybrid peptide.” Thesemolecules were synthesized in both orientations to assess the effect ofN- or C-terminal locations on the ability of the IFBM to bind tocollagen or BMP-2.

To determine if these IFBM's increased the amount of BMP retained by acollagen sponge, we mixed the IFBM with BMP, added the mixture to asponge, allowed them to bind for 1.5 hours, washed the sponge anddetected the bound BMP with anti-BMP antibodies. Briefly, stock IFBMsolutions were prepared by weighing 1-2 mg peptide and solubilizing inwater. The final peptide concentration was determined by analyzing thepeptide absorbance at 280 nm and the extinction coefficient. For eachrow, 20 μL of peptide were added to each well of a polypropylenemicrotiter plate. BMP was then added to each of these wells in athreefold dilution series, starting with 32 μM BMP. The IFBM and BMPwere allowed to mix at room temperature for 30 minutes.

To each well, a 2/16″ diameter collagen sponge (Medtronic) was added.The collagen and peptide solutions were allowed to incubate for 1.5hours at room temperature. Sponges were then rinsed three times with 200μL Medtronic buffer at 2200 rpm for 1 minute. To each sponge, a primaryantibody directed at BMP (diluted 1:1000; R&D Systems #MAB3552) wasadded for 1 hour at room temperature. A secondary antibody conjugated toalkaline phosphatase (1:5000) was then incubated in the system for 0.5hour at room temperature. PNPP was used to develop the system andabsorbances were read at 405 nm. Results are shown in FIG. 5.

The results shown in FIG. 5 demonstrate that IFBM AFF7010 retains moreBMP on the sponge than the sponges without IFBM. IFBM AFF7008 andAFF7017 increase the amount of BMP on the sponge when compared to noIFBM, but to a lesser extent than AFF7010. The increased retention ofBMP to the sponge is not seen by adding AFF2006, a BMP-binding peptidethat does not contain a collagen-binding sequence.

To show that this effect is dose dependent not only on the amount of BMPput onto the sponge but also on the amount of IFBM present, a series oftwo-dimensional dose response curves was obtained in which theconcentrations of both the IFBM and BMP were varied. These results areshown in the FIGS. 6A-6D and demonstrate that the binding of BMP to thecollagen sponge is dependent on both BMP concentration and IFBMconcentration. Increasing the concentration of the IFBM (AFF7005,AFF7006, AFF7009, or AFF7010) leads to a larger amount of BMP-2 that isretained on the collagen.

Example 5 Peptides that Bind to Stainless Steel

Selection of stainless steel-binding peptides was performed as describedabove for the titanium-binding peptides except that 5/32 inch stainlesssteel beads were used instead of titanium beads. The stainless steelbinding peptides that were isolated are shown in Table 8. In someembodiments, an exemplary binding module of the invention comprises onlythat portion of the sequence shown in uppercase letters.

TABLE 8 Stainless Steel Binding Peptides Phage SEQ ID DesignationPeptide Sequence NO: AP08-03 ssSSYFNLGLVKHNHVRHHDSsr 39 AP08-02ssCHDHSNKYLKSWKHQQNCsr 40 AP08-01 ssSCKHDSEFIKKHVHAVKKCsr 41 AP08-04ssSCHHLKHNTHKESKMHHECsr 42 AP08-06 ssVNKMNRLWEPLsr 43

Example 6 Peptides that Bind to Teflon

Selection of Teflon (GoreTex®; polytetrafuorethylene (PTFE))-bindingpeptides was performed as described above for the titanium-bindingpeptides except that sections of GoreTex fabric were used instead oftitanium beads. The Teflon-binding peptides that were isolated are shownin Table 9. In some embodiments, an exemplary binding module of theinvention comprises only that portion of the sequence shown in uppercaseletters.

TABLE 9 Teflon Binding Peptides Clone SEQ. ID. Number Peptide SequenceNO.: AP16-01 ssCWSRFRLFMLFCMFYLVSsr 95 AP16-02 srCIKYPFLYCCLLSLFLFSsr 96

Example 7 Isolation of Peptides that Specifically Bind to BMPRI and/orBMPRII

Identification of peptides that bind to BMPRI and/or BMPRII: In order toidentify peptides that specifically bind to Bone Morphogenic ProteinReceptor I (BMPRIA) and/or Bone Morphogenic Protein Receptor II(“BMPRII”), phage display libraries are screened to identify phageencoding peptides that bind to the extracellular domains of eachreceptor. The extracellular domains of these receptors are known in theart (Rosenweig et al. (1995) Proc. Nat'l. Acad. Sci. USA 92: 7632-7636;Ten Dijke et al. (1994) J. Biol. Chem. 269: 16985-16988). Various phagelibraries are screened. Where appropriate, a phage library can beselected that is designed around a specific amino acid motif or that wasmade with a particular amino acid bias. BMPRIA and BMPRII (R&D Systems,Cat. Nos. 315-BR/CF and 811-BR) are dissolved in carbonate coatingbuffer (100 mM NaHCO₃, pH 9.6); 100 μl of this solution is added to thewells of a 96-well Immulon®-4 microtiter plate (Dynex Technologies,Chantilly, Va.). The plate is incubated overnight at 4° C. and then thenonspecific binding sites on the surface of the polystyrene are blockedwith 1% Bovine Serum Albumin (BSA) in carbonate coating buffer. Theplate is then incubated for an hour at room temperature with shaking at50 rpm. The wells are then washed 5 times with 300 μl of PBS-T (SigmaChemical Co., St. Louis, Mo., Cat. #P-3563). Each library is diluted inPBS-T and added at a concentration of 1010 pfu/ml in a total volume of100 ul. The plates are then incubated at room temperature with shakingat 50 rpm for 3 hours; unbound phage is then removed with 5 washes ofPBS-T. Bound phage are recovered by denaturation with 0.1 M glycinebuffer pH 2.2 (see Phage Display of Peptides and Proteins: A LaboratoryManual, 1996, eds. Kay et al. (Academic Press, San Diego, Calif.)).Eluted phage are neutralized with phosphate buffer and then added to E.coli DH5α cells in 2×YT media. This mixture is incubated overnight at37° C. in a shaker at 210 rpm. Phage supernatant is harvested bycentrifuging at 8500×g for 10 minutes. Second and third rounds ofselection are performed similarly to the first round of selection usingthe phage from the previous round of selection as the input phage. Phagedisplay techniques are well known in the art, for example, as describedin Sparks et al. (1996) “Screening phage-displayed random peptidelibraries,” pp. 227-253 in Phage Display of Peptides and Proteins: ALaboratory Manual, eds. Kay et al. (Academic Press, San Diego, Calif.).

To identify phage that specifically bind to BMPRIA or BMPRII,conventional ELISAs are performed using an anti-M13 phage antibodyconjugated to horseradish peroxidase (HRP), followed by the addition ofchromogenic agent ABTS (Sigma Chemical Co., St. Louis, Mo., Cat.#A3219). Relative binding strengths of the phage are determined bytesting serial dilutions of the phage for binding to BMP receptors in anELISA. The DNA encoding each selected peptide is isolated and sequencedto determine the amino acid sequence of the selected peptide.

These peptides are then linked together to create an analyte module thatwill bind to each of BMPRI and BMPRII, forming a heterodimer of thesetwo receptors so as to induce signaling. Candidate peptides aresynthesized and biotinylated and their binding to the BMP receptorsconfirmed. Briefly, the biotinylated peptides are synthesized with alinker between the BMP receptor binding sequence and the attached biotinmoiety. This linker has the amino acid sequence GSSGK, which serves toseparate the biotin moiety from the receptor binding portion of thepeptide and which is flexible. Peptides are synthesized usingsolid-phase peptide synthetic techniques on a Rainin Symphony PeptideSynthesizer (Rainin Instrument Co., Emeryville, Calif.) using standardFmoc chemistry. N-α-Fmoc-amino acids (with orthogonal side chainprotecting groups) can be purchased from Novabiochem(Calbiochem-Novabiochem, Laufelfingen, Switzerland). After all residuesare coupled, simultaneous cleavage and side chain deprotection will beachieved by treatment of a trifluoroacetic acid (TFA) cocktail. Crudepeptide is precipitated with cold diethyl ether and purified byhigh-performance liquid chromatography on a ShimadzuAnalytical/Semi-preparative HPLC unit on a Vydac C18 silica column(preparative 10 μm, 250 mm×22 mm; Grace Vydac Co., Hesperia, Calif.)using a linear gradient of water/acetonitrile containing 0.1% TFA.Homogeneity of the synthetic peptides is evaluated by analytical RP-HPLC(Vydac C18 silica column, 10 μm, 250 mm×4.6 mm) and the identity of thepeptides is confirmed with MALDI-TOF-MS, for example, as performedcommercially at the UNC-CH Proteomics Core Facility.

Generation of peptides that bind to BMPRI and/or BMPRII with highaffinity: Peptides that are initially identified as binding to BMPRIand/or BMPRII may have low binding affinities, e.g., in the mid- tolow-μM range, whereas it may be preferable that peptides for use in anIFBM have higher binding affinities, e.g., in the nM range. To identifysuch peptides, libraries of variants of the initially identifiedpeptides are constructed and screened by affinity selection againstBMPRI and/or BMPRII.

Determination of binding affinity is evaluated using procedures known inthe art. For example, BMPRI, BMPRII, and appropriate control proteinsare dissolved in carbonate coating buffer (100 mM NaHCO₃, pH 9.6) andadded to the wells of a 96-well polypropylene plate. After incubationovernight at 4° C., the wells are blocked with 1% BSA in PBS-T. Eachreceptor and control is tested for binding over a range of peptideconcentrations from 0 to 200 μM in sterile PBS (pH 7.2). The wells arethen washed to remove unbound peptide and a streptavidin-alkalinephosphatase conjugate solution (SA-AP) from USB (United StatesBiochemical #11687) is added to each well to quantify the amount ofbound peptide. Streptavidin-alkaline phosphatase activity is measuredusing the chromogenic reagent p-nitrophenyl phosphate reagent(Sigma-Aldrich, Inc., SigmaFast tablets, catalog #N1891) and measuringabsorbance at 405 nm. To determine a binding curve and rough K_(D),absorbance is plotted as a function of the concentration for eachpeptide. The impact of other factors on binding can be assessed, such asfor example, pH, temperature, salt concentration, buffer components, andincubation time.

To create and identify peptides that bind to BMPRI and/or BMPRII withhigher affinity, phage libraries are created based on an amino acidmotif identified among the initial peptides isolated as binding to BMPRIand/or BMPRII and screened further for peptides with improved bindingproperties. Such techniques are known in the art (see, for example,Hyde-DeRuyscher et al. (2000) Chem. Biol. 7: 17-25; Dalby et al. (2000)Protein Sci. 9: 2366-2376).

Characterization of agonist activity of hybrid peptides comprisingBMPRI-binding peptides and BMPRII-binding peptides: Synthetic peptidesare chemically synthesized that comprise both a BMPRI-binding peptideand a BMPRII-binding peptide connected with a flexible linker (e.g., alinker having the sequence GSSGSSG). Alternatively, the tworeceptor-binding peptides may be linked through the α and ε amino groupsof a lysine (e.g., as in Cwirla et al. (1997) Science 276: 1696-1699 orin Wrighton et al. (1997) Nat. Biotechnol. 15: 1261-1265). Thesepeptides are about 40 amino acids in length and are readily synthesizedand purified.

These peptides are then assayed for BMP activity such as, for example,the induction of alkaline phosphatase activity in mouse mesenchymalC3H10T1/2 cells as known in the art and described, for example, by Chenget al. (2003) J. Bone Joint Surg. Am. 85-A: 1544-1552 and Ruppert et al.(1996) Eur. J. Biochem. 237: 295-302. Briefly, C3H10T1/2 cells are addedto a 96-well plate (3×10⁴ cells per well in a volume of 200 μl) inGibco® MEM/EBSS medium (Invitrogen Corp., Carlsbad, Calif., Cat#11095-080) with 10% FBS and appropriate antibiotics and antimycotics.Cells are permitted to adhere to the plate for at least 3 hours byincubating at 37° C. in a 5% CO₂ atmosphere. Media is then asepticallyaspirated and BMP-2 or peptides are added at various concentrations inhigh-glucose Gibco® DMEM (Invitrogen Corp., Carlsbad, Calif., Cat.#11965-092) plus 2% FBS. Cells are incubated with the tested compoundsfor three days, at which time the media is aspirated and the cells arewashed three times with 300 μl of PBS (Gibco® PBS, Cat. #14190-144,Invitrogen Corp., Carlsbad, Calif.). 100 μl of pNPP (p-NitrophenylPhosphate Sigma Fast Tablet Set Cat #N-1891) in H₂O is added to eachwell and the color is allowed to develop for up to 18 hours at 37° C.before absorbance is read at 405 nm.

EC₅₀ values are then determined using methods known in the art. TypicalEC₅₀ values for this assay for BMP-2 range between 1 μg/ml and 10 μg/ml(see, e.g., Wiemann et al. (2002) J. Biomed. Mater. Res. 62: 119-127).It is known in the art that BMP-2 isolated from different sources canshow different levels of activity, and one of skill in the art canadjust procedures accordingly to take these differences into account toachieve the desired result. For example, it is known in the art thatrecombinant human BMP-2 (“rhBMP-2”) prepared using CHO cells hasactivity which differs 5-10 fold from the activity of recombinant humanBMP-2 prepared using E. coli (see, e.g., Zhao and Chen (2002),“Expression of rhBMP-2 in Escherichia coli and Its Activity in InducingBone Formation,” in Advances in Skeletal Reconstruction Using BoneMorphogenic Proteins, ed. T. S. Lindholm).

Immobilization of hybrid peptides onto collagen: Hybrid peptides thatshow BMP activity are synthetically linked to a peptide that binds tocollagen. Briefly, a peptide containing the collagen binding module andthe BMPRI-binding module is synthesized with an orthogonal protectinggroup on an amino acid in the linker between the modules, such asFmoc-Lys(Dde)-OH. The Dde protecting group on the c amino group of thelysine side chain can be selectively removed and a BMPRII-bindingpeptide coupled to the c amino group. Alternatively, a linear peptidecan be synthesized that comprises the collagen-binding module, theBMPRI-binding module, and the BMPRII-binding module.

The collagen-bound hybrid peptide is then tested for its BMP activity,such as by assaying for the induction of alkaline phosphatase activityin mouse mesenchymal C3H10T1/2 cells while the hybrid peptide is boundto a collagen matrix. Briefly, 5-mm disks of collagen are washed withPBS and added to the cell-based BMP activity assay.

Example 8 Sterilization of Surfaces Coated with IFBMs

IFBM-coated surfaces were treated with electron-beam sterilizationprocedures and gamma sterilization procedures. The binding performanceof the coated surfaces was assessed before and after the sterilizationprocedures. Assays were performed on polystyrene and titanium surfaces.For the polystyrene assay, a binding module (“AFF-0002-PS”) wasbiotinylated and relative binding was assessed by exposing the bindingmodule to streptavidin-conjugated alkaline phosphatase. The resultsshowed that the amount of biotinylated peptide that was bound to thepolystyrene surface was essentially identical before and after thesterilization procedures. Similar results were obtained for an assay ofa binding module (“AFF-0006-Ti”) on titanium; in this assay, theperformance of the coated surface before sterilization was approximatelyequal to its performance after sterilization.

Example 9 Preliminary Toxicity Testing

A PEGylated polystyrene-binding peptide was coated onto variouspolystyrene surfaces and tested as follows for adverse effects includingcytotoxicity, hemolysis, and coagulation. The procedures were performedin Albino Swiss Mice (Mus musculus). As further discussed below, none ofthe IFBMs tested showed any signs of toxicity.

To assay for acute systemic toxicity, polystyrene squares (each square4×4 cm; a total of 60 cm²) were incubated for 70-74 hours at 37° C. in20 mL of one of two vehicles: 0.9% USP normal saline or cotton seed oil(National Formulary). Five mice were each injected systemically witheither vehicle or vehicle-extract at a dose rate of 50 mL extract per kgbody weight. Mice were observed for signs of toxicity immediately afterinjection and at 4, 24, 48, and 72 hours post-injection. None of theanimals injected with the vehicle-extract showed a greater biologicalreaction than those that received vehicle alone.

Coated surfaces were assayed for partial thromboblastin time accordingto ISO procedure 10993-4 (International Organization forStandardization, Geneva, Switzerland). Briefly, fresh whole human bloodwas drawn into vacutainer tubes containing sodium citrate and were spundown to isolate plasma, which was stored on ice until use. Coatedpolystyrene squares (as described above) were then incubated in theplasma at a ratio of 4 cm² per 1 mL for 15 minutes at 37° C. inpolypropylene tubes and agitated at 60 rpm. The plasma extract was thenseparated, placed on ice, and tested on a Cascade® M-4 manual hemostasisanalyzer (Helena Laboratories, Beaumont, Tex.). Clotting time was notsignificantly different than that observed for pure plasma or thestandard reference control.

Cytotoxicity was assayed in L-929 Mouse Fibroblast Cells as specified inISO 10993-5. Briefly, 60.8 cm² of polystyrene-coated squares wasextracted into 20.3 mL of Eagle's Minimum Essential Medium+5% FBS at 37°C. for 24 hours. Positive, negative and intermediate cell-line testdishes were incubated at 37° C. in a humidified 5% CO₂ atmosphere.Cultures were evaluated for cytotoxic effects by microscopic observationat 24, 48, and 72 hours. The positive control showed a strong cytotoxicreaction score of “4” while test cells maintained a healthy (“0” score)appearance across all time points (score of “0”). Intermediate controlcells scored as “2” across all time points.

Hemolysis testing measures the ability of a material or material extractto cause red blood cells to rupture. The test performed was ASTM F-756Direct Contact Method. Saline was used to extract leachable substances.Coated polystyrene surface was extracted and then added to citratedrabbit blood (3.2%, diluted with PBS to obtain a total blood hemoglobinconcentration of 10 mg/ml). A score of 0.4% was observed which fallsinto the passing category of 0-2%. The negative control returned a scoreof 0.1% and the positive control returned a score of 12.2%.

1-19. (canceled)
 20. An implant comprising a polypeptide that binds to abone morphogenetic protein, wherein the polypeptide is selected from thegroup consisting of: (i) the polypeptide set forth in any one of SEQ IDNOs: 27 or 73; (ii) the polypeptide set forth in any one of SEQ ID NOs:11-24, 44-72, or 74; (iii) the polypeptide having at least 70% identityto any one of the polypeptides set forth in SEQ ID NOs: 11-24, 44-55,57-72, or 74; (iv) the polypeptide having at least 75% identity to anyone of the polypeptides set forth in SEQ ID NOs: 11-24, 44-72, or 74;and (v) the polypeptide that is a conservatively substituted variant ofany one of the polypeptides set forth in SEQ ID NOs: 11-24, 44-72, or74.
 21. The implant of claim 20, wherein the polypeptide is set forth inany one of SEQ ID NOs: 27 or 73 and the polypeptide consists of no morethan 40 amino acids in length.
 22. The implant of claim 20, wherein thepolypeptide is set forth in any one of SEQ ID NOs: 27 or 73 and thepolypeptide consists of no more than 30 amino acids in length.
 23. Theimplant of claim 20, wherein the polypeptide is set forth in any one ofSEQ ID NOs: 27 or 73 and the polypeptide consists of no more than 20amino acids in length.
 24. The implant of claim 20, wherein thepolypeptide is set forth in any one of SEQ ID NOs: 27 or 73 and thepolypeptide consists of no more than 17 amino acids in length.
 25. Theimplant of claim 20, wherein the polypeptide is set forth in any one ofSEQ ID NOs: 27 or 73 and the polypeptide consists of no more than 10amino acids in length.
 26. The implant of claim 20, wherein thepolypeptide is set forth in any one of SEQ ID NOs: 27 or 73 and thepolypeptide consists of no more than 7 amino acids in length.
 27. Theimplant of claim 20, wherein the bone morphogenetic protein is bonemorphogenetic protein-2.
 28. A method for promoting bone formation, themethod comprising placing a polypeptide that binds to a bonemorphogenetic protein (BMP) at a site for bone formation, wherein thepolypeptide binds and presents the BMP to promote the bone formation,and wherein the polypeptide is selected from the group consisting of:(i) the polypeptide set forth in any one of SEQ ID NOs: 27 or 73; (ii)the polypeptide set forth in any one of SEQ ID NOs: 11-24, 44-72, or 74;(iii) the polypeptide having at least 70% identity to any one of thepolypeptides set forth in SEQ ID NOs: 11-24, 44-55, 57-72, or 74; (iv)the polypeptide having at least 75% identity to any one of thepolypeptides set forth in SEQ ID NOs: 11-24, 44-72, or 74; and (v) thepolypeptide that is a conservatively substituted variant of any one ofthe polypeptides set forth in SEQ ID NOs: 11-24, 44-72, or
 74. 29. Themethod of claim 28, wherein the bone morphogenetic protein is bonemorphogenetic protein-2.
 30. A method for promoting osteointegration,and/or accelerating healing, and/or reducing inflammation at a site ofan implant, the method comprising localizing a polypeptide that binds toa bone morphogenetic protein (BMP) at the site of the implant, whereinthe polypeptide binds and presents the BMP for the promotedosteointegration, and/or accelerated healing, and/or reducedinflammation, and wherein the polypeptide is selected from the groupconsisting of: (i) the polypeptide set forth in any one of SEQ ID NOs:27 or 73; (ii) the polypeptide set forth in any one of SEQ ID NOs:11-24, 44-72, or 74; (iii) the polypeptide having at least 70% identityto any one of the polypeptides set forth in SEQ ID NOs: 11-24, 44-55,57-72, or 74; (iv) the polypeptide having at least 75% identity to anyone of the polypeptides set forth in SEQ ID NOs: 11-24, 44-72, or 74;and (v) the polypeptide that is a conservatively substituted variant ofany one of the polypeptides set forth in SEQ ID NOs: 11-24, 44-72, or74.
 31. The method of claim 30, wherein the bone morphogenetic proteinis bone morphogenetic protein-2.
 32. A method for binding bonemorphogenetic protein (BMP) in vitro, the method comprising incubating aBMP with a polypeptide that binds to the BMP under conditions suitablefor binding, wherein the polypeptide is selected from the groupconsisting of: (i) the polypeptide set forth in any one of SEQ ID NOs:27 or 73; (ii) the polypeptide set forth in any one of SEQ ID NOs:11-24, 44-72, or 74; (iii) the polypeptide having at least 70% identityto any one of the polypeptides set forth in SEQ ID NOs: 11-24, 44-55,57-72, or 74; (iv) the polypeptide having at least 75% identity to anyone of the polypeptides set forth in SEQ ID NOs: 11-24, 44-72, or 74;and (v) the polypeptide that is a conservatively substituted variant ofany one of the polypeptides set forth in SEQ ID NOs: 11-24, 44-72, or74.
 33. The method of claim 32, wherein the bone morphogenetic proteinis bone morphogenetic protein-2.